Genetic dissection of cis-acting elements controlling DNA replication timing and genome architecture

控制 DNA 复制时间和基因组结构的顺式作用元件的遗传解剖

• 批准号: 10327671
• 负责人: Jesse Lloyd Turner
• 金额: $ 4.06万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327671
• 关键词: 3-Dimensional; Address; Architecture; Binding Sites; Biological; Biological Assay; CCCTC-binding factor; Chromatin; Chromatin Loop; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Replication Timing; DNA Structure; DNA biosynthesis; Data; Development; Disease; Dissection; Elements; Enhancers; Eukaryota; Foundations; Genetic; Genetic Transcription; Genome; Genomics; Goals; Hi-C; Histone Acetylation; Histones; Individual; Knock-out; Length; Link; Location; Mission; Molecular; Mus; Nuclear; Outcome; Play; Process; Public Health; Publishing; Regulation; Regulatory Element; Replication Origin; Research; Role; S Phase; Site; Structure; System; Time; Transcription Initiation Site; United States National Institutes of Health; Work; base; cell type; chromatin remodeling; chromosome conformation capture; cis acting element; cohesin; embryonic stem cell; experimental study; fundamental research; genome integrity; innovation; insight; novel; pluripotency; pluripotency factor; programs; spatiotemporal; transcription factor

PROJECT SUMMARY/ABSTRACT DNA replication is essential for the structural and functional integrity of genomes, and spatiotemporal control of DNA replication (replication timing, RT) is intimately related to 3D genome architecture and organization, however, we know little about how it is regulated. The lab has recently identified cis-acting elements that are crucial for controlling RT, chromosome architecture, and transcription in mouse embryonic stem cells (mESCs), termed early replication control elements (ERCEs). ERCEs contain many active chromatin marks, such as acetylated histone residues and are co-occupied by pluripotency factors Oct4, Sox2, and Nanog (OSN). ERCEs also contain transcription start sites (TSSs), which may account for their role in regulating transcription. ERCEs form 3D interactions independent of architectural proteins CTCF and cohesin. What is unknown is whether ERCEs are one functional unit that coordinately regulates RT, architecture, and transcription, or if they are composed of discrete, separable elements that independently regulate different nuclear functions. The hypothesis is that ERCEs are composed of multiple elements, such as OSN binding sites and TSSs, that can be uncoupled, to determine their individual roles in regulating RT, architecture, and transcription. The rationale is that RT, genome architecture, and transcription are all commonly disrupted in disease states, and so understanding how they are regulated will lead to a better understanding of the molecular and cellular basis of disease. AIM 1 will use CRISPR to genetically dissect ERCEs in order to identify the sequences harboring activity necessary for ERCE activity, such as RT and transcription. AIM 2 will transfer ERCEs or components of ERCEs to ectopic sites and assay their sufficiency to promote early replication and transcription and to alter chromatin architecture at the ectopic genomic location. This contribution will be significant because it will address the regulation of DNA structure and function and how that regulation is perturbed in disease. The proposed research is innovative because the discovery of ERCEs provide novel biological questions and approaches to investigate the regulation of chromosome structure and function.

项目总结/摘要 DNA复制对于基因组的结构和功能完整性以及基因组的时空控制至关重要。 DNA复制（复制时间，RT）与3D基因组结构和组织密切相关， 然而，我们对它是如何受到监管的知之甚少。该实验室最近发现了顺式作用元件， 对于控制小鼠胚胎干细胞（mESC）中的RT、染色体结构和转录至关重要， 早期复制控制元件（ERCE）。ERCE含有许多活性染色质标记，例如 乙酰化组蛋白残基，并被多能性因子Oct 4、Sox 2和Nanog（OSN）共同占据。ERCEs 也含有转录起始位点（TSS），这可能是它们在调节转录中的作用。ERCEs 形成独立于结构蛋白CTCF和粘附素的3D相互作用。不知道的是， ERCE是协调调节RT、结构和转录的一个功能单位，或者如果它们是 由独立调节不同核功能的离散的、可分离的元件组成。的 假设ERCE由多个元件组成，例如OSN结合位点和TSS，其可以 解偶联，以确定它们在调节RT，架构和转录中的各自作用。其基本原理是 RT、基因组结构和转录在疾病状态下通常都被破坏， 了解它们是如何调节的，将有助于更好地了解细胞内的分子和细胞基础。 疾病AIM 1将使用CRISPR对ERCE进行遗传解剖，以确定具有活性的序列 ERCE活性所必需的，如RT和转录。AIM 2将转移ERCE或ERCE组件 异位位点，并测定其是否足以促进早期复制和转录，并改变染色质 在异位基因组位置的结构。这一贡献将是重要的，因为它将解决 DNA结构和功能的调节以及这种调节在疾病中是如何被扰乱的。拟议研究 是创新的，因为ERCE的发现提供了新的生物学问题和研究方法 染色体结构和功能的调节。